P-014: USE OF OXYGEN GRADIENT EKTACYTOMETRY TO MEASURE EFFICACY IN CELL-BASED THERAPIES
Authors: KANNE C., JOHN T., GURKAN U., SHEEHAN V.
Journal: HemaSphere (2022)

Purpose: The degree of red cell phenotype correction, and therefore cure, in gene-based therapy and allogeneic hematopoietic stem cell transplant (allo-HSCT) with mixed donor chimerism (MDC), may not be captured by conventional laboratory tests like a complete blood count or hemoglobin profile. We propose to define a cure as the achievement of at least the sickle cell trait level of red blood cell (RBC) deformability and sickling potential. RBC deformability is markedly abnormal in SCD; these abnormalities contribute to vaso-occlusion, ischemia, and SCD complications Even fully oxygenated, sickle RBCs are less deformable than those of HbAA or HbAS individuals; upon deoxygenation, deformability further declines. To determine if the rheology of blood from SCD patients post-cell-based therapy and post-HSCT with varying degrees of donor chimerism fall into the HbSS range of values, we functionally assessed the peripheral blood from a series of 7 post alloHCT SCD patients and one post-gene addition autologous hematopoietic stem cell transplant subject using oxygen gradient ektacytometry. Materials and methods: Peripheral blood samples (EDTA) from6 SCD patients post-allo-HSCT, one matched sibling donor sample, and a peripheral blood sample from an individual treated with Bluebird Bio’s Group C lentivirus-based gene addition were collected and immediately analyzed using oxygen gradient ektacytometry (LORRCA). Oxygen gradient ektacytometry measures RBC deformability (elongation index, EI), under a range of pO2 (150-0 mmHg). EImax is the deformability of the oxygenated sickle RBC; EImin is the deformability of deoxygenated RBCs. The point of sickling (PoS) is the pO2 at which HbS polymerizes. Additional clinical data including Hb profile, donor chimerism, and symptoms were obtained via chart review. Reference ranges for genotypes were generated as described above using n=45 HbSS samples ages 2-21, on hydroxyurea, chronic transfusion, or untreated; n=14 HbAS, and n=43 HbAA. Results: 6/7 allo-HSCT subjects exhibited normal or trait level rheology as measured by oxygen gradient ektacytometry, based on their donor genotype; one patient with 40% whole blood chimerism and an HbS less than 50% had EImin, EImax, and PoS values in the HbSS disease range. The gene addition treated subject had an HbS of 40%, yet their EImin, EI max and PoS also fell within the disease range. Both subjects with disease range RBC rheology reported SCD symptoms post-cell-based therapy, namely pain events. The other subjects did not report SCD symptomatology post-cell-based therapy. Conclusion: Oxygen gradient ektacytometry identified uncorrected, SCD level RBC dysfunction in two subjects post-cell-based therapies with conventional laboratory test values consistent with a cure. The current post-HCT evaluation depends on chimerism and hemoglobin profiles and would not detect the significant functional abnormalities visible by Lorrca with Oxygenscan biomarkers that indicate that Patient 2 is at risk for SCD related complications. Determination of a cure in gene-based therapies is an area of active research. Our results suggest that functional RBC analysis is needed for the management of post alloHCT SCD patients and may be even be more essential to assessing new gene-based therapy approaches to curing patients with SCD.Oxygen gradient ektacytometry analysis of an allo-HSCT subject with 40% donor chimerism and their HbAA matched sibling donor (blue line).Oxygen gradient ektacytometry analysis of a subject post gene addition based gene therapy, multiple measurements. T. JOHN declares a conflict of interest: Research support/Scientific studies: HGB-210 site PI (bluebird bio)